通过常压室温等离子体诱变和基因组复制工程辅助连续进化技术提升酿酒酵母琥珀酸产量及其发酵优化OA

The kokumi taste of soy sauce is closely related to its quality.Succinic acid is one of the key compounds enhancing this at-tribute.This study aimed to improve the yield of succinic acid by a yeast strain via non-genetically engineered microbial breeding technolo-gies and to optimize the fermentation process,thereby providing technical support for enhancing the kokumi quality of soy sauce.Firstly,the physicochemical properties and sensory characteristics of commercial soy sauce were analyzed and a significant positive correlation be-tween succinic acid concentration and kokumi intensity was observed.Then,a combinational strategy of atmospheric and room temperature plasma(ARTP)mutagenesis and genome replication engineering assisted continuous evolution(GREACE)was applied to improve the succinic acid yield using Saccharomyces cerevisiae LA-FR as start strain.Through ARTP mutagenesis,the mutant strain LA-FR-100 was obtained and further subjected to GREACE evolution combined with high-salinity stresses.A genetically stable evolved strain LA-FR-100-15 with a succinic acid yield of 620.12 mg/L was obtained,which was 106％higher than that of start strain.Finally,the fermentation conditions for succinic acid production by the evolved strain were optimized.The optimal medium composition was as follows,glucose 80 g/L,yeast extract 13 g/L,corn steep liquor 7 g/L,K2HPO4·3H2O 3 g/L,MgSO4·7H2O 1.5 g/L and ZnSO4·7H2O 0.5 g/L.The optimal fermentation parameters were 26 ℃,initial pH 6.5,and 9％inoculum.Under the optimal fermentation condition,the yield of succinic acid by LA-FR-100-15 strain reached 1.54 g/L,which was 148.7％higher than that before optimization.Through fed-batch fer-mentation in 5 L fermenter,the final succinic acid yield reached 9.6 g/L.The results obtained in this study delivered a non-genetically engineered microbial solution and an optimized process to enhance the kokumi of soy sauce.